This invention relates to the preparation and purification of lactase.
Lactase, or beta-galactosidase, is an enzyme effective for the hydrolysis of lactose to glucose and galactose. Accordingly, lactase is useful in the dairy industry, for example in cheese making, where the addition of lactase to the cheese milk increases the rate of lactose hydrolysis and thereby shortens cheese ripening times, increases production capability and allows production of improved products. Lactase is also useful for administration to persons who are physiologically intolerant to lactose.
Lactase is an intracellular constituent of certain microorganisms which are readily produced by conventional fermentation methods, including for example microorganisms of the genus Kluyveromyces, previously considered as Saccharomyces, especially Kluyveromyces fragilis and Kluyveromyces lactis, and other yeasts such as those of the genera Candida, Torula and Torulopis, and the like. In the past, cells of such microorganisms have been cultivated in a suitable nutrient medium, harvested and dried, whereafter the whole dried cells are used to impart lactase activity in the desired application. This method is undesirable in that the other cell constituents are also added to the cheese milk and can impart undesirable taste and odor characteristics. Prior art methods to overcome this problem have involved methods of releasing the lactase from the interior of the cells, especially by breaking the cell walls by mechanical homogenization, grinding or chemical means. However, these methods generally require long processing times, are expensive and will release not only lactase but other intracellular enzymes and proteins, and their degration products, from which the lactase must desirably be separated. In this regard, one particular problem has been that in the release of intracellular lactase from microorganism cells, the enzyme protease is also released and it has been difficult to remove the protease activity from the lactase preparation. When protease is present in the lactase preparation added to cheese milk to accelerate cheese ripening, undesirable bitter off-flavors, attributed to peptide formation by proteolytic degradation of milk proteins, may be produced. Accordingly, in view of the above there has been a need for improved processes for the release of lactase from microorganisms and there has further been a need for processes for the purification of lactase preparations so obtained, especially for processes to selectively remove protease activity from lactase preparations.